Use of triplex structure dna in transferring nucleotide sequences

ABSTRACT

A method to induce an immune response in a host in need thereof, comprises administering to the host, recombinant lentiviral vector particles comprising:
         a) a GAG polypeptide or a functional GAG-polypeptide derivative;   b) a POL polypeptide or a functional POL-polypeptide derivative ;   c) an ENV polypeptide or a functional ENV-polypeptide derivative; and   d) a recombinant polynucleotide.
 
The recombinant polynucleotide comprises a transgene placed under the control of regulatory signals for transcription and expression, regulatory signals, of lentiviral origin, for reverse transcription, expression and packaging, and a polynucleotide comprising a cis-acting central initiation region (cPPT) and a cis-acting termination region (CTS). The regions are of lentiviral origin and are inserted in a functional orientation with the regulatory signals of lentiviral origin. The polynucleotide forms a DNA triplex during reverse transcription.

The present application relates to the use of DNA sequences which arecapable of having a triple-stranded structure or organisation (known astriplex DNA) for transferring nucleotide sequences into cells, and torecombinant vectors containing such triplex sequences.

Thus the invention concerns the definition and provision of novel meanswhich can be used, for example, in the context of protocols for genetherapy or transgenesis for the production of transgenic animals orplants or recombinant cells or cell lines. Such means comprise producingnovel vectors which can transfer a nucleotide sequence, in particular asequence of therapeutic interest, into target cells in the human oranimal body.

An important limitation to current gene therapy approaches lies in thevectorisation of the gene of therapeutic interest. Retroviral vectorsderived from an oncovirus, principally from MoMLV, have been widely usedfor gene transfer. Their application is largely limited by the fact thatoncoviruses only integrate into target cells which are activelydividing. In contrast, lentivirus have the unique capacity amongretroviruses of infecting differentiated non mitotic cells and representviral candidates of interest for the development of novel vectors. Whileretaining the advantages of an oncoviral vector (absence ofimmunogenicity, stable integration), lentiviruses could enable in vivotransduction of non mitotic differentiated tissues (brain, muscle,liver, lung . . . ) and could therefore have a wide range ofapplications in gene therapy.

Different attempts at constructing retroviral vectors from lentiviruseshave been reported. In this respect, the work of Poznansky M. et al (J.Virol 1991, 65, 532-6), Naldini et al (Science, 1996, 272, p 263-7)carried out using the HIV retrovirus and the work of Poeschla E M et al(Nature Medicine, 1998, 4, p 354-7) carried out using the FIV retroviruscan be cited.

The inventors have searched the determinants involved in the mechanismof entry of the retrovirus genome into infected cell nuclei (nuclearimport mechanism).

The identification of a triplex DNA determinant essential for import hasled the inventors to define novel means, and in particular vectors, foruse in transferring genes, or more generally sequences of nucleotides(henceforth termed “transgenes”), into target cells. In particular, theinventors have worked from the HIV (human immunodeficiency virus)retrovirus, a member of the lentivirus family, and have identified andisolated a viral determinant responsible for the nuclear import ofproviral DNA of HIV into target cells: central triplex DNA. This DNAtriplex has been shown to be able to function in vectors out of thenatural context of the HIV-1 genome, as a nuclear import determinantenabling the vector genome to enter the nucleus of target cells.

Mechanisms for retroviral DNA entry into the nucleus exhibitconsiderable differences from one retroviral family to another. Thelentivirus genome is capable of crossing the nuclear membrane of theinterphasic nucleus by addressing followed by translocation of itspre-integration complex (linear DNA and associated proteins) through thenuclear pore. Thus such viruses are capable of replicating in theabsence of division of the target cell. In particular, they infectdifferentiated tissue macrophages and dendritic cells, cells at the coreof the transmission, dissemination, and the physiopathology of HIV. Incontrast, oncovirus genomes and spumavirus genomes are incapable ofcrossing the barrier constituted by the nuclear membrane. Theirpre-integration complex must await mitosis and disorganisation of thenuclear membrane in order to accede to the mitotic chromosomes and beintegrated.

The viral determinants responsible for nuclear import of the DNA of theHIV-1 virus have been studied by the inventors. The identification andfunctional comprehension of the molecular mechanisms of nuclear importof the HIV pre-integration complex is of fundamental importance. Theinventors have identified an original mechanism for nuclear import ofthe HIV-1 genome by which this import is governed by a DNA structure, atriplex at the centre of linear DNA molecules, generated by stepsparticular to lentiviral reverse transcription.

The triplex DNA structure present at the centre of linear DNA moleculesgenerated during lentiviral reverse transcription, in particular in theHIV retrovirus, has been described by the inventors in different priorpublications (Charneau P. et al., J. Mol. Biol. 1994, 241, 651-662;Charneau P et al, Journal of Virology, May 1991, p 2415-2421; CharneauP. et al., Journal of Virology, 1992, vol. 66, p 2814-2820).

The DNA structure forming a triplex during viral reverse transcriptionis a polynucleotide comprising a cis-acting central initiation region,or polypurine tract (cPPT), and a cis-acting termination region (CTS),these regions enabling initiation of transcription of a +strand thesynthesis of which is initiated by the PPT region present in the centreof the HIV genome or other lentiviruses, and interruption of synthesisof a second +strand the synthesis of which is initiated at a 3′ PPT siteupstream of the retroviral LTR (FIG. 1).

Formation of the triplex DNA structure is the consequence of a discretestrand displacement event in the retrovirus genome, blocked by the CTSsequence (Charneau P. et al., J. Mol. Biol., 1994).

It should be understood that the term “triplex DNA” used here designatesa triple-stranded region of DNA, with no reference to the structure ofthose strands (free displaced strand, or forming a triple helix or aD-loop, etc . . . . ).

The structure of the DNA triplex formed during reverse transcriptionenables or at least contributes to the entry of the retroviral genomeinto the cell nucleus, thus allowing infection of non mitotic cells.

Starting from the identification of this required mechanism for entry ofthe retrovirus into the nucleus of target cells, the inventors haveproduced a novel generation of lentiviral vector, including the triplexDNA region. The introduction of a DNA fragment from the HIV-1 genomecomprising the cPPT and CTS sequences which are cis-acting into an HIVvector system increases transduction of genes into the cells bystimulating the amount of nuclear import of the vector DNA. Thisgeneration of lentiviral triplex vectors considerably improvestransduction of the gene into the cells whether or not they are mitotic.

The invention concerns a nucleotide sequence of retroviral orretroviral-like origin, which can be prepared synthetically, comprisingcPPT and CTS regions which are cis-acting in reverse transcription ingeneral, and in particular two associated polynucleotides when they areplaced in the normal retroviral genome, each polynucleotide containingat least 10 nucleotides.

The nucleotide sequence of the invention (see FIG. 11G where thecis-acting sequences of interest are boxed) comprises on one side, ashort nucleotide sequence termed “cPPT” in the case of HIV-1 (minimum 10base pairs) and on the other side, a sequence termed “CTS” of at least10 base pairs in the case of HIV-1. The two cis-acting sequences and anucleotide sequence from a retroviral genome located between these twocis sequences correspond to about 120 nucleotides in the case of thenatural HIV-1 genome.

The invention also concerns a nucleotide sequence comprising three DNAstrands constituted by, on one hand, the CTS region (or an equivalentregion in the case where the origin of the genome used is other thanHIV-1 but with the same properties as the CTS region published byCharneau et al., 3. Mol. Biol., 1994) and, on the other hand, upstreamof the CTS, a region containing about 90 to 110 nucleotides, preferably99 nucleotides in the case of HIV-1.

The invention concerns a polynucleotide comprising a double stranded DNAfragment corresponding to the cPPT (polypurine tract) region associatedwith a polynucleotide sequence naturally present in the HIV-1 genome (oran equivalent natural or synthetic sequence), and finally a CTSnucleotide region which adopts a conformation which defines the end ofthe triple stranded region (3′ end) after reverse transcription.

This triple-stranded conformation is termed a “triplex sequence”.

By way of example, the triplex sequence is that shown in FIG. 11F forHIV-1 or FIG. 11G. In vivo, when present in a vector for use forpenetrating the nuclear membranes of eukaryotic cells, the triplexstimulates import of DNA into the nucleus of the cell to be modified ortransduced.

The invention concerns the use of this triplex sequence alone or in avector to introduce nucleotide sequences to which the triplex sequenceis bound into the nucleus of the receiving eukaryotic cell.

Thus the invention provides a recombinant vector, characterized in thatit comprises a polynucleotide containing a cis-acting central initiationregion (cPPT) and a cis-acting termination region (CTS), these regionsbeing of retroviral or retroviral-like origin, said vector furthercomprising a defined sequence of nucleotides (transgene or the sequenceof interest) and regulatory signals for reverse transcription,expression and packaging of retroviral or retroviral-like origin.

The term “polynucleotide” used here refers to any nucleic acid sequencein the form of a single or double or triple strand, whether DNA, forexample cDNA, or RNA.

By way of example, the invention concerns the transfer of transgenes fortherapeutic purposes, in particular within the context of somatic genetherapy protocols, to insert a nucleotide sequence which modulates orrepairs a defective activity in the somatic cells of an organism torectify poor function of an endogenous gene, or to enable expression ofa supplementary function, in particular a function which suppresses theexpression or the activity of a gene, for therapeutic purposes.

The expression “therapeutic” as used here means the search for orproduction of a preventative or curative effect, or the search for orproduction of an improvement or stabilisation of the pathological stateof a patient.

Within the context of the invention, and by way of example, thenucleotide sequences termed transgenes or nucleotide sequences ofinterest can be genes or gene portions or sequences derived from genes,for example cDNA or RNA. They may also be antisense sequences, negativemutant sequences of a given gene, or sequences involved in the functionsof gene transcription, expression or activation, or sequences suitablefor activation of prodrugs or cytotoxic substances.

The activity of the transgene sequences of the invention can also be tostimulate or induce an immune, cellular or humoral response, for examplewhen used to transform cells presenting an antigen.

Thus the invention can be applied to the preparation of vectors used forgene therapy in various domains such as that of hereditary diseasescomprising altering a gene, these diseases including Duchenne's musculardystrophy, cystic fibrosis, neurodegenerative diseases or acquireddiseases such as malignant diseases naturally leading to a weak responseof the immune system. The invention can also envisage immunotherapytreatments to stimulate the response to pathogenic agents, for exampleby the production of CTL, for example in the case of diseases such ascancers or diseases such as AIDS, or to reduce the response against selfantigens in the case of autoimmune diseases.

The invention also concerns the provision of means for producingimmunogenic compositions or prophylactic or therapeutic vaccines, orimmunogenic compositions.

Lentiviral vectors containing a DNA triplex of the invention are alsoused to construct transgenic animals by transduction of genes into celllines or embryonic cells.

The vector of the invention contains a transgene inserted under thecontrol of viral or non viral sequences regulating transcription orexpression.

The transgene can be included in an expression cassette comprisingsuitable sequences for regulating its expression in the cell.

A first particularly interesting embodiment of the invention is that inwhich the recombinant vector is characterized in that the sequences ofretroviral origin it contains are derived from the genome of alentivirus.

Within the context of the present application, the term “derivative”encompasses any sequence identical to the sequence contained in thegenome of the retrovirus, or any sequence modified by mutation,insertion, deletion or recombination, provided that it preserves theessential function it possesses in the retroviral genome, with regard toits insertion into the vector of the invention.

Such a sequence could be obtained by any known means enablingidentification and isolation of sequences of nucleotides from theirorganism of origin, in particular comprising the steps of cloning and/oramplification, or by synthesis using any known technique.

Alternatively, the vector of the invention is characterized in that thesequences of retroviral-like origin are derived from a retrotransposon.The retrotransposon yeast TY1 can be mentioned in this regard (Heyman Tet al).

The recombinant vector thus described can, for example, be a plasmidrecombined by a retroviral or retroviral-like construction and atransgene, if necessary contained in an expression cassette.

The recombinant vector can also be a retrotransposon, a phage, such as aλ phage or a filamentary phage which can be introduced into bacteria, ora vector capable of transforming yeasts such as a YAC.

Such a vector can be used for cell transduction, and in particularpackaging of cells and/or target cells, by any method which is known perse, including transfection or infection or transduction, for example byan adenovirus or AAV type vector containing the triplex lentiviralvector.

A vector as defined above can be transcomplemented by one or moreadditional vectors carrying sequences coding for structure polypeptidesfrom the genome of a selected retrovirus, in particular a lentivirus, orstructure polypeptides of a retrotransposon.

In this regard, the vector of the invention can be transcomplemented byproviding sequences coding for the polypeptides GAG, POL and ENV, or fora portion of these polypeptides sufficient to enable formation ofretroviral particles aimed to vectorise the recombinant vector deprivedof viral genes and comprising the transgene the expression of which issought.

A vector of the invention can be characterized in that the transgene orsequence of interest is contained in an expression cassette comprisingsignals regulating transcription and expression.

In general, the vector(s) used for transcomplementation into retroviralor retroviral-like proteins are depleted in packaging signals.

In this regard, the vectors prepared using the techniques of Goldman etal (1997) for use in transcomplementation of a recombinant vector of theinvention can be cited.

The invention also concerns recombinant retroviral vector particlescomprising:

-   -   a) a gas polypeptide corresponding to nucleproteins of a        lentivirus or to functional polypeptide derivatives (GAG        polypeptides);    -   b) a pol polypeptide constituted by the proteins RT, PRO, IN of        a lentivirus or a functional polypeptide derivative (POL        polypeptide);    -   c) an envelope polypeptide or functional polypeptide derivatives        (ENV polypeptides);    -   d) a recombinant nucleotide sequence comprising a defined        nucleotide sequence (transgene or a sequence of interest) placed        under the control of regulatory signals for transcription and        expression, a sequence containing regulatory signals for reverse        transcription, expression and packaging of retroviral or        retroviral-like origin and a polynucleotide comprising a        cis-acting central initiation region (cPPT) and a cis-acting        termination region (CTS), said regions being of retroviral or        retroviral-like origin and being inserted in a functional        orientation with said regulatory signals of retroviral or        retroviral-like origin.

The invention also concerns recombinant retroviral vector particlescomprising:

-   -   a) a nucleotide sequence termed a gag sequence coding for        nucleoproteins of a lentivirus or for functional polypeptide        derivatives (GAG polypeptides);    -   b) a nucleotide sequence termed a pol sequence coding for the        proteins RT, PRO, IN and RN of a lentivirus or for a functional        polypeptide derivative (POL polypeptide);    -   c) regulatory signals for transcription and expression of the        gag and pol sequences;    -   d) a nucleotide sequence termed an env sequence coding for        envelope polypeptides or for functional polypeptide derivatives        (ENV polypeptides), the env sequence being placed under the        control of regulatory signals for transcription and expression;    -   e) a recombinant nucleotide sequence comprising a defined        sequence of nucleotides (transgene), placed under the control of        regulatory signals for transcription and expression, a sequence        containing regulatory signals for reverse transcription,        expression and packaging of retroviral or retroviral-like        origin, and a polynucleotide comprising a cis-acting central        initiation region (cPPT) and a cis-acting termination region        (CTS), said regions being of retroviral or retroviral-like        origin, said regions being inserted in a functional orientation        with regulatory signals of retroviral or retroviral-like origin.

In one variation, the invention provides a nucleotide sequencecomprising a polynucleotide comprising a cis-acting central initiationregion (cPPT) and a cis-acting termination region (CTS), of retroviralor retroviral-like origin, each of said two regions flanking aconcatenation of internal nucleotides, said cis-acting cPPT and CTSregions being inserted into said nucleotide sequence, in a functionalorientation with regulatory signals for reverse transcription ofretroviral or retroviral-like origin.

GAG and POL polypeptides are nucleoprotein polypeptides from precursorscleaved by viral protease. POL polypeptides comprise reversetranscriptase (RT), protease (PRO), integrase (IN) and Rnase H (RN) ofthe retrovirus. If necessary, other retroviral proteins are also used toconstruct vector particles. It should be noted that the terms “proteins”or “polypeptides” used here encompass the non glycosylated forms or theglycosylated forms of the polypeptides in question.

The gag, pol and env sequences used to construct retroviral vectorparticles can, if necessary, be modified by mutation, for example bypoint mutation or by deletion or insertion of one or more nucleotides,or may originate from recombinant chimeras originating from differentretroviruses, for example HIV-1 and HIV-2 or HIV-1 and CAEV (CaprineArthritis Encephalitis Virus), provided that they allow the productionof functional polypeptides for the production of viral particles capableof vectorising the transgene to be expressed. In particular, mutatedsequences are used to increase the safety of the retrovirus produced.

Advantageously, the recombinant vector of the invention or recombinantvector particles are such that the transgene is under the control ofregulatory signals for transcription and expression of non retroviralorigin. An example of a promoter which can be used to control expressionof the transgene is the CMV promoter, the PGK promoter or EF1α promoterdescribed by Tripathy, S K et al (PNAS 1994, 91, p 11557-11561).

In a variation of the invention, the transgene can be placed under thecontrol of regulating signals previously identified as being retroviralor retroviral-like in origin, in particular under the control of the LTRsequence.

A lentivirus used to derive the retroviral construction of the inventioncan be selected from the HIV retrovirus, for example HIV-1, HIV-2 or anydifferent isolate of these two types, or for example from the CAEV(Caprine Arthritis Encephalitis Virus) virus, EIAV (Equine InfectiousAnaemia Virus), VISNA, SIV (Simian Immunodeficiency Virus) or FIV(Feline Immunodeficiency Virus).

A particularly advantageous vector of the invention is a vectorcharacterized in that the polynucleotide is a DNA sequence comprisingthe cis-acting central initiation region (cPPT) and the terminationregion (CTS) of the genome of an HV1 retrovirus or any other lentivirus.

The central PPT sequence or cPPT sequence is a relatively conservedsequence in lentiviruses and is identified by the presence of numerouspurine residues certain of which are shown in FIG. 11H. Mutations, evenpoint mutations in one of these regions, can destroy the functionalnature linked to the formation of DNA triplex structures.

The identification of cPPT sequences is facilitated by the fact that apolypurine sequence located at the upstream edge (5′) of the 3′ LTR inall retroviruses is repeated in the centre of the genome inlentiviruses. This cPPT sequence can be an exact repeat as in the HIV-1virus, or slightly modified in other lentivirus (FIG. 11H). The centraltermination sequence CTS has been characterized for the HIV-1 virus(Charneau et al, 1994). It is located about one hundred nucleotidesdownstream of the cPPT sequence. In other lentiviruses, CTS sequencecandidates are also about a hundred nucleotides (80 to 120 nucleotides)downstream of the cPPT sequence. The probable position of the CTSsequence is indicated for several lentiviruses in FIGS. 11A to 11E.

The CTS sequence of the EIAV lentivirus has recently been characterized(Scott R. Stetor et al Biochemistry 1999, p 3656-67). According to theauthors, in EIAV, the cPPT and CTS sequences are respectively

5′ AAC AAA GGG AGG GA 3′ and 5′ AAA AAA TTT TGT TTT TAC AAA ATC 3′.

Examples of preferred polynucleotides for use in the invention which canbe cited are the sequences shown in FIG. 11, more precisely thesequences between the two regions cPPT and CTS, including the sequencesin those regions.

If necessary, the sequence of nucleotides comprising cPPT, the internalpolynucleotide (i.e., binding the cPPT to the CTS sequence) and the CTSsequence can be point mutated or mutated by deleting or insertingnucleotides. By way of example, point mutations have been produced inthe cPPT sequence of HIV-1 and have shown that it retained residualinfectivity in the cells (Charneau et al, J. Virol. 1992, 66, p2814-2820).

The invention encompasses any mutated sequence for cPPT or CTS which isat least 60% identical to the natural homologous cis-acting nucleotidesequence from which it originates. In the case of chimeral cis-actingsequences, the percentage is applied to each mutated nucleotide sequenceof the chimera.

Modifications to the nucleotide sequence of the PPT or cPPT or CTSregions can be introduced to construct the triplex DNA of the invention.Such modifications can reach up to 40% of the natural sequence.

The identity of the nucleotide sequences which vary with respect to thenatural sequences is calculated strictly with respect to the cPPT or CTSindividually and not with respect to the complete triplex DNA nucleotidesequence.

The region between the cPPT and CTS is constituted by a polynucleotidewhich can either be that found in the original retroviral genome betweenthe CTS and PPT or it can be different therefrom provided that thetriplex DNA retains its properties as regards nuclear import of thepolynucleotide to enable the nucleotide sequence of interest to be takeninside the nucleus.

The polynucleotide of the invention can be introduced into a replicativeor non replicative vector. In the case of a retroviral vector, it is anon replicative vector.

In order to prepare large quantities of retroviral vector particles, itis possible to use adenoviral type vectors into which the polynucleotidecorresponding to the retroviral genome which contains the triplex DNAsequences and those of the gag, pol and env genes has been introduced.

These adenoviral vectors can optionally be rendered replicative byintroducing an origin of replication sequence.

FIG. 11G shows the cPPT and CTS sequences of HIV-1 in boxes.

In all cases, mutated sequences will be used which retain the capacityto form a DNA triplex during reverse transcription of the genome in thetarget cell.

A recombinant vector in accordance with a particular implementation ofthe invention can thus comprise all or a portion of the retroviral orretrotransposon LTR sequences, retroviral PBS sites, and 3′-terminalPPT, the retroviral sequence necessary for packaging of the vectorgenome in the vector particle. The LTR sequence can be partiallydeleted, in particular in the U3 region.

A particular vector of the invention is the plasmid pTRIP.EGFP which hasbeen deposited at the CNCM (Collection National de Cultures deMicroorganismes [National Micro-organism Culture Collection] by theInstitut Pasteur, France) on 15 Apr. 1998, accession number I-2005. Therestriction map for this vector is shown in FIG. 10.

Another vector in accordance with the invention is the plasmidpTRIP.MEL-IRES-GFP deposited at the CNCM on 20 Apr. 1999, accessionnumber I-2185. This vector is the plasmid pTRIP.MEL-IRES-GFP shown inFIG. 14.

A particular recombinant vector of the invention is characterized inthat the gag, pol and env sequences are also derived from lentivirussequences, in particular an HIV retrovirus, more particularly HIV-1 orHIV-2.

In a further implementation of the invention, the gag and pol sequencesare derived from an HIV retrovirus and the env sequence is derived froma retrovirus which is distinct from HIV or from a virus, for example thevesicular somatitis virus (VSV).

In general, and as a function of the expression of the transgene whichis being researched, an env sequence coding for env polypeptides whichare amphotropic with respect to the host in which the transgene is to beexpressed can be selected, or env sequences coding for ecotropic envpolypeptides can be selected. The tropism of the env sequence can be aspecifically human tropism.

The invention also provides recombinant vector particles comprising arecombinant sequence of nucleotides comprising a defined nucleotidesequence (transgene or sequence of interest) placed under the control ofregulatory signals for transcription and expression, regulatory signalsfor reverse transcription and expression, a sequence containingregulatory signals for expression and packaging and a polynucleotidecomprising a cis-acting central initiation region (cPPT) and acis-acting terminal region (CTS).

The invention also provides recombinant vector particles comprising asequence of recombinant nucleotides comprising a defined nucleotidesequence (transgene) placed under the control of regulatory signals fortranscription and expression, regulatory signals for reversetranscription, expression and packaging of a retrotransposon and apolynucleotide comprising a cis-acting central initiation region (cPPT)and a cis-acting terminal region (CTS), these regions being derived froma retrotransposon and inserted in a functional orientation withretrotransposon regulatory signals.

Further, the invention also concerns recombinant vector particlescomprising:

-   -   a) a GAG polypeptide corresponding to the nucleoproteins of a        retrotransposon or to functional polypeptide derivatives;    -   b) a POL polypeptide corresponding to the RT, PRO, IN proteins        of a retrotransposon or to a functional polypeptide derivative;    -   c) regulatory signals for transcription and expression of gag        and pol sequences;    -   d) a recombinant nucleotide sequence comprising a defined        nucleotide sequence (transgene) placed under the control of        regulatory signals for reverse transcription, expression and        packaging of a retrotransposon and a polynucleotide comprising a        cis-acting central initiation region (cPPT) and a cis-acting        terminal region (CTS), said regions being derived from a        retrotransposon and inserted in a functional orientation with        retrotransposon signal regulators.

Further, the invention concerns recombinant vector particles resultingfrom expression of

-   -   a) a nucleotide sequence termed a gag sequence coding for        nucleproteins of a retrotransposon or for functional polypeptide        derivatives (GAGpolypeptides);    -   b) a nucleotide sequence termed a pol sequence coding for the        RT, PRO and IN proteins of a retrotransposon or for a functional        polypeptide derivative (POL polypeptide);    -   c) regulatory signals for transcription and expression of gas        and pol sequences, said particles comprising a recombinant        sequence of nucleotides comprising a defined sequence of        nucleotides (transgene) placed under the control of regulatory        signals for transcription and expression, a sequence containing        regulatory signals for reverse transcription, expression and        packaging of a retrotransposon and a polynucleotide comprising a        cis-acting central initiation region (cPPT) and a cis-acting        termination region (CTS), these regions being derived from a        retrotransposon and inserted in a functional orientation with        retrotransposon signal regulators.

The invention further concerns recombinant retroviral-like particlescomprising:

-   -   a) a polynucleotide comprising a cis-acting central initiation        region (cPPT) and a cis-acting termination region (CTS), said        regions being derived from a retrotransposon and inserted in a        functional orientation with retrotransposon signal regulators;    -   b) a polypeptide corresponding to nucleoproteins of a        retrotransposon or to functional polypeptide derivatives        (GAGpolypeptides);    -   c) a pol polypeptide corresponding to the RT, PRO, IN proteins        of a retrotransposon or to a functional polypeptide derivative        (POL polypeptide);    -   d) a viral envelope polypeptide;    -   e) a recombinant nucleotide sequence comprising a defined        sequence of nucleotides (transgene or sequence of interest)        placed under the control of regulatory signals for transcription        and expression, regulatory signals for reverse transcription,        expression and packaging of a retrotransposon.

As an example, the invention concerns a recombinant vector as definedabove, in which the regulatory signals for reverse transcription,expression and packaging and the polynucleotide comprising the cPPT andCTS regions are derived from a retrotransposon, for example a yeastretrotransposon.

In general, the signals regulating transcription and expression of thetransgene or sequences coding for structure polypeptides of the vectorparticle, when they are not retroviral or retroviral-like in origin, areadvantageously inducible or conditional signals which are capable ofleading to tissue-specific expression.

Recombinant cells characterized in that they are recombined with avector according to any one of the above definitions are alsoencompassed by the invention. Recombination can be carried out using anysuitable means, in particular transfection or infection, especiallytransfection or transduction by a vector.

The cells can thus be transiently or stably transfected. They may bepackaging cells or target cells, in particular cells in which atherapeutic effect is sought by expression of the transgene.

Particularly interestingly, recombinant cells which are capable ofexpressing the transgene due to transduction using a vector of theinvention are non mitotic differentiated eukaryotic cells.

The invention also encompasses the preparation of recombinant nonmitotic primary eukaryotic cells, or mitotic cells.

Examples which can be cited are the cells of the lung, brain, epithelialcells, astrocytes, microglia, oligodendrocytes and neurons, musclecells, hepatic cells, dendritic cells, neuron cells, bone marrow cells,macrophages, fibroblasts, lymphocytes and haematopoietic cells.

Thus the invention relates to compositions with a therapeutic purpose,characterized in that they comprise a vector as described above, or arecombinant cell defined as indicated above.

The invention also concerns an immunogenic composition comprising avector as described above or recombinant cells as defined above, saidcomposition being capable of leading _(t)o an immune, cellular orhumoral response in a given host.

The invention thus provides a polynucleotide as defined above comprisingretroviral or retroviral-like cPPT and CTS regions which provides accessto its use for nuclear import of a nucleotide sequence (transgene), inparticular ex vivo in defined cells,

Further, the invention provides a polynucleotide as defined aboveassociated with a nucleotide sequence of interest or with a transgene.

Finally, the invention concerns the use of a polynucleotide comprising acis-acting central initiation region and a cis-acting termination region(CTS), these regions being retroviral or retroviral-like in origin, fortransfection or transduction of eukaryotic cells with a transgene orpolynucleotide of interest.

It also concerns the use of a recombinant vector or a polynucleotide ofthe invention for in vivo transduction.

Further characteristics and advantages of the invention will becomeapparent from the following examples and figures.

LEGEND TO FIGURES

FIG. 1: Reverse Transcription of lentivirus

Reverse transcription of lentiviral genomes differs from that ofoncogenic retroviruses by the synthesis of the +strand in two distincthalves. A downstream segment is initiated at a central copy of thepolypurine tract (cPPT) characteristic of lentiviral genomes. Synthesisof the upstream +strand is terminated after displacement of the discretestrand at the centre of the genome. Blocking of displacement of thestrand by reverse transcriptase is governed by a cis-acting sequence ofthe HIV genome: the CTS (central termination sequence). The finalproduct of reverse transcription of the lentivirus is a linear DNAcarrying a central triple-stranded DNA structure (central triplex) overa length of about one hundred nucleotides.

FIG. 2: Plasmids Used for Producing HIV Vector Particles

Vector particles were produced by co-transfection of three plasmids: thevector plasmid comprising (pTRIP) or not comprising (pHR) the cis-actingsequences responsible for triplex formation, a packaging plasmidproviding, trans, the structural proteins and enzymes of the particle(pCMVΔR8.2 or pCMVΔR8.91) Naldini et al, 1996 and Zufferey et al, 1997),and a VSV virus envelope expression plasmid (VSV-G).

Only the pertinent parts of the plasmids co-transfected into HeLa cellsare shown (Naldini et al PNAS, October 1996, Zufferey et al, NatureBiotech, 1997).

The packaging plasmids pCMVΔR8.2 or pCMVΔR8.91 enable expression of theproteins from gag and pol.

PMD.G codes for the heterologous VSV envelope. The vector plasmidspHR-TRIP were derived from the pHR'CMVlacZ plasmid (Naldini et al): awild type or mutant triplex sequence has been inserted and the lacZreporter gene, changed or otherwise in EGFP.

FIG. 3: Impact of Triplex on Transduction of EGFP into HeLa Cells

HeLa cells, cultivated in an 8 chamber Labtek, were transduced bydifferent vectors expressing the autofluorescent protein EGFP. Theinfections were normalised for the quantity of capsid protein (P24 ELISAkit, Dupont) to 2 ng of P24 per inoculum. 48 hours post-infection, thecells were fixed with 1% PBS PFA, mounted in mowiol, then observed witha fluorescence microscope. Three independent fields are shown for thevector of origin with no triplex (HR.EGFP, top), for the vector withtriplex (TRIP.EGFP, middle) or for a vector containing a mutated nonfunctional triplex sequence (TRIP D.EGFP, bottom). The right hand sideshows the different transductions in the presence of nevirapine, aninhibitor for HIV-1 reverse transcriptase.

FIG. 4: Quantification of the Degree of Transduction of the EGFP Gene byHIV Vectors with or without Triplex

HeLa cells transduced by 2 ng P24 of EGFP vectors with or withouttriplex were trypsined 48 hours post-infection. The percentages of cellswhich were positive for EGFP expression were calculated by flowcytometry (FITC channel). In all cases, transduction was inhibited inthe presence of nevirapine, an HIV-1 reverse transcriptase inhibitor. InFIG. 4C, the presence of the triplex DNA was observed in the vectorstimulated by transduction of GFP (or another gene of interest) in cellsin mitosis or non mitotic cells. This transduction was multiplied by afactor of 20 with respect to the results obtained with vectors without atriplex sequence (for example, see Naldini et al, Science, 1996).

FIG. 5: Quantification of the Degree of Transduction of the LacZ Gene byHIV Vectors with r without Triplex

The impact of the triplex on transduction was calculated by infectingHeLa cells, cultivated in 96 well trays, using different vectorsexpressing the lacZ reporter gene. 48 hours post infection, the culturetrays were lysed and the beta-galactosidase activity was measured usinga luminescent reaction kit (Boehringer). Each transduction was carriedout in triplicate with an innoculum normalised to 2 ng of P24.

Upper panel: proliferating HeLa cells.

Lower panel: HeLa cells blocked in their cycle by aphidicoline.

Transduction of the lacZ gene was multiplied by a factor of 6 with avector containing a triplex sequence with respect to a vector with notriplex sequence.

FIG. 6 a & b: Impact of Triplex on ex vivo Transduction of the EGFP Genein Rat Spinal Primary Cells

Primary explant cells from rat spinal cord were infected with 300 ng ofP24 for each vector with and without triplex, and observed using afluorescence microscope as described above.

FIG. 7: Impact of Triplex on in vivo Transduction of EGFP Gene andLuciferase Gene in Rat Brain

FIG. 7-a-1: Transduction at Injection Site.

The EGFP gene was transferred by direct injection into the striatum ofthe rat brain of 2 microlitres of the vector corresponding to 50 ng ofP24.

Observation of the sections under fluorescence microscopy showed a largetransduction of EGFP in the presence of triplex (left hand panel) andvery little without (right hand panel).

FIG. 7-a-2: Another section representing the experiment described above.

FIG. 7-b: Quantification of Impact of Triplex on in vivo Transduction inthe Brain.

FIG. 7-b-1: Impact of triplex on transduction of the gene coding forluciferase in in vitro HeLa cells. The graph shows the luciferaseproduction quantified by measuring luminescence (Promega® kit). Thepresence of the triplex in the vector increased transduction of theluciferase gene by a factor of 8.

FIG. 7-b-2: In vivo quantification of luciferase activity in rat brainsafter injection of vectors coding for luciferase, with or withouttriplex. The presence of triplex stimulates luciferase transduction by afactor of 8.

FIG. 7-b-3: Same experiment as 7-b-2 but carried out in the mouse.

FIG. 8: Strategy for Analysis of Amount of Nuclear Import of Vector DNA.

A quantitative test enabling the reverse transcription, nuclear importand integration or circularisation kinetics of the vector DNA intransduced cells was developed. This test advantageously replaceddetection by PCR amplification of circles with two LTRs, markers fornuclear import of viral DNA into the nucleus of the infected cell(Bukrinsky et al, Nature 1993, 365, p 666-669). The non integratedlinear vector DNA, circular DNAs with one or two LTRs and integratedvector DNA were detected by Southern blot and quantified using aPhosphorimager using the following restriction digestion strategy: thetotal DNA of the transduced cells was digested with EcoNI and AvaII (twounique sites in the vector genome) then hybridised with a DNA probegenerated by PCR precisely spanning the EcoNI site. This probe reactedwith different fragments: the internal 0.77 kb fragment, common to allof the vector DNA forms, and for which quantification using thephosphorimager indicated the total quantity of reverse transcribedvector DNA; a distal fragment of 1.16 Kb specifically indicating thequantity of linear non integrated DNA. After supplemental digestion bythe Xhol enzyme, spots with one or two LTRs appeared at 1.4 Kb and 2 Kbrespectively. The quantity of integrated vector DNA was calculated bysubtracting the signals corresponding to non integrated vector DNA,linear DNA and spots from the signal corresponding to the total reversetranscribed DNA. In the case of a lack of nuclear import, the expectedvector DNA profile in the transduced cells is an accumulation of nonintegrated linear DNA. In contrast, if the vector DNA reaches thenuclear compartment of the cell, the essential part of the linear DNA isintegrated into the cellular chromatin and circularises.

FIG. 9 a: Analysis of Nuclear Import of Vector DNA

Southern blot analysis 48 hours post transduction in HeLa cells showed atypical lack of nuclear import in the case of the vector without triplex(HR GFP) or containing the triplex sequence in the reverse orientation,which is non functional (TRIP, GFP inv). In the case of these vectors,the signal corresponding to non integrated linear DNA was equivalent tothe total DNA signal, indicating that the essential part of the vectorDNA remained in the linear form instead of becoming integrated. In thecase of the TRIP.GFP vector, the intensity of the signal correspondingto linear DNA was very much lower than the total DNA signal, indicatingthat a large fraction of vector DNA had been imported into the nucleusand had been integrated therein.

FIG. 9-b: Kinetic analysis of degree of nuclear import of vector DNAswith triplex (TRIP-GFP) or without triplex (HR-GFP, TRIPinv-GFP).

FIG. 9-c: Quantification of state of vector DNA in transduced cells.

Phosphorimager quantification of the Southern blot of FIG. 9 b showedthat 48 hours post-transduction, the majority of the DNA of the vectorswithout triplex were in the form of linear non integrated DNA; only alittle vector DNA had integrated or circularised. The triplex-freevectors (HR-GFP and TRIPinv-GFP) exhibited a typical lack of nuclearimport. In contrast, in the case of the TRIP-GFP vector, more than 60%of the DNA had integrated into the genome of the transduced cell andonly a little vector DNA subsisted in the form of non integrated linearDNA. Introduction of the triplex sequence into the vector hadcomplemented the lack of nuclear import of the HR-GFP vector to thelevel of the wild type. In fact, the vector DNA profile obtained in thecase of the TRIP-GFP vector was comparable with that of a wild typeHIV-1 virus. This result shows that the triplex sequence is the onlydeterminant of nuclear import lacking in the HR-GFP construction. Onlythe integrated form of the DNA vector was active.

FIG. 10: Restriction Map for pTRIP.EGFP Vector.

FIGS. 11A-11F: Polynucleotide Sequence Comprising cPPT and CTS Regionsof the CAEV, EIAV, VISNA, SIV_(AGM), HIV-2_(ROD) and HIV-I_(LA1)viruses.

FIG. 11G: represents the triplex DNA sequence of the HIV-1 virus. Thecis-acting regions, cPPT and CTS, are boxed and printed in boldcapitals.

FIG. 11H: represents the alignment of cPPT and 3′ PPT sequences inseveral lentiviruses. The top line corresponds to the 3′ PPT sequencepresent in all retroviruses upstream of 3′ LTR. The bottom linecorresponds to the internal repetition of the PPT sequence termed thecPPT in lentivirus.

FIG. 12:

FIG. 12 represents the production of CTL in vitro from human dendriticcells transduced by the triplex vector with a melanoma CTL polyepitopeconstituted by epitopes the sequences of which are described in FIG. 15as the gene of interest.

These dendritic cells were brought into contact with mononuclear cells(PBLo). The CTL activity was measured after re-stimulation by thecorresponding antigenic peptides.

The effective cells/target cells ratio is shown along the abscissa.

FIG. 13: Cytotoxic response after immunising mice with theTRIP.MEL-IRES-GFP vector.

FIG. 14: Restriction map for the pTRIP.MEL-IRES-GFP vector.

The E. coli strain containing the pTRIP.MEL-IRES-GFP vector wasdeposited on 20 Apr. 1999 at CNCM, accession number I-2185.

FIG. 15: Sequences for specific CT1 HLA A2.1 melanoma epitopes includedin the polyepitopic construction of the pTRIP.MEL-IRES-GFP vector. Thesequences of the polyepitope are underlined to distinguish each epitope.

FIG. 16: Very High Efficiency Transduction of CD34+ Stem Cells byTriplex HIV Vectors.

Flow cytometry (FACS) analysis of transduction of the GFP gene inhaematopoietic CD34+ stem cells by the TRIP-GFP vector. The percentageof CD34+ cells transduced by the TRIP-GFP vector was more than 85%. Thisefficiency was notably more efficient than the degree of transductionobtained previously with an HIV vector with no triplex (HR-GFP) in CD34+cells (Miyoshi H et al, Science 1999, 283, p 682-6).

METHOD AND APPARATUS Construction of Plasmid Vectors:

The pTRIP-LacZ and pTRIP-EGFP plasmids derive from the pHR'CMVlacZconstruction (Naldini et al, 1996). The lacZ reporter gene ofpHR'CMVlacZ was replaced by the ORF of the autofluorescent protein EGFP.The EGFP gene was amplified by PCR from the pEGFP-N1 plasmid (Clontech)using thermostable Pfu polymerase (Stratagene).

The sequences for the PCR primers used were as follows:

Bam EGFP: 5′ cc gga tcc cca ccg gtc gcc acc 3′

Xho EGFP: 5′ cc ctc gag cta gag tcg cgg ccg 3′

PCR amplification was carried out for 30 cycles under the followingconditions:

-   -   denaturation 95° C., 30 sec;    -   hybridisation 50° C., 1 min;    -   elongation 75° C., 30 sec.

The BamHI and XhoI restriction sites were added at the 5′ and 3′ endrespectively of the EGFP PCR fragment so as to insert it in anorientated manner into the pHR'CMV vector fragment, itself digested withBamHI and XhoI. Insertion of the EGFP PCR fragment using conventionalrecombinant DNA techniques (Maniatis et al, 1983) generated the pHR-EGFPplasmid.

A 184 by fragment corresponding to the central region of the HIV-1genome and comprising the cis-acting cPPT and CTS regions responsiblefor the formation of the triplex during reverse transcription of the HIVwas inserted in the Clal site of the pHR-EGFP and pHR'CMVlacZ plasmids,upstream of the CMV promoter. The central triplex region was amplifiedby PCR from complete proviral plasmids of the LAI HIV-1 genomecomprising the wild type triplex sequence (pBRU3; Charneau et al, 1991),mutated in the cis-acting termination sequence CTS (pCTS; Charneau etal, 1994) or mutated in the cis-acting central initiation sequence cPPT(p225; Charneau et al, 1992).

The sequences for the PCR primers were as follows:

Nar/Eco TRIP+: 5′ gtc gtc ggc gcc gaa ttc aca aat ggc agt att cat cc 3′Nar TRIP−: 5′ gtc gtc ggc gcc cca aag tgg atc tct gct gtc c 3′

The PCR reaction conditions were identical to those described above.

The PCR triplex fragments, digested by Nan were inserted into the ClaIsite of the pHR GFP and pHR'CMV lacZ plasmids by competitive T4 DNAligase/ClaI ligation/digestion to eliminate the self re-circularisedvector during the ligation step. The orientation of the insertion wasanalysed by Xhol/EcoRI digestion, the EcoRI having been introduced intothe 5′ Nar TRIP+ PCR primer.

The resulting plasmids were termed pTRIP.EGFP in the correct orientationfor the triplex and pTRIPinv.EGFP in the reverse, non functionalorientation. The vectors comprising a mutated version of the triplexwere termed pTRIP X.EGFP, X corresponding to the code of the startingmutant virus (AG, D, CTS or 225) (Charneau et al, J. Mol. Biol. 1994,Charneau et al, J. Viral 1992). Starting from the different plasmidspTRIP.EGFP or pTRIP X.EGFP, the EGFP gene was replaced by lacZ byorientated XhoI/BamHI exchange. The resulting plasmids were respectivelytermed pTRIP.Z, pTRIPinv.Z, pTRIP CTS.Z, pTRIP 225.Z.

Constructions of HR luc and TRIP luc Vectors

The BamHI-EGFP-XhoI fragment of the RH GFP and TRIP GFP vectors wasreplaced by the BamHI-Luc-XhoI fragment of the pGEM-luc plasmid(Promega) coding for luciferase.

Production of Non Infectious Vector Particles

HIV vectors were produced using a modification of the protocol describedby Naldini et al, 1996. The vector particles were produced by transientco-transfection with calcium phosphate of human 293T cells (ATCC),cultivated in a DMEM (ICN), 10% FCS, penicillin, streptomycin medium.Semi confluent 175 cm² boxes were simultaneously transfected by threeplasmids:

-   -   15 μg of plasmid coding for the envelope of the vesicular        stomatis virus (VSV), pMD.G (Naldini et al, 1996);    -   30 μg of packaging plasmid, pCMVΔR8.2 (Naldini et al, 1996) or        pCMVΔR8.91 (Zufferey et al, 1997);    -   and 30 μg of the different plasmid vectors pHR or pTRIP.

The calcium phosphate/DNA co-precipitates were left in contact with thecells for 24 hours, the medium was then collected every 24 hours up topost transfection day 3. The cellular debris of the vector supernatantswas eliminated by low speed centrifugation. The vector supernatants werestored at −80° C.

Concentration of Vector Particles

The use of the very stable VSV-G envelope to pseudotype the vectorparticles enabled them to be concentrated by centrifugation. Thesupernatant vectors, collected as described above, were ultracentrifugedin 30 ml conical bottom tubes (Beckman) for 90 min at 17000 rpm at +4°C. with a SW 28 rotor (Beckman). The pellets were then taken up in 190†μl of PBS, centrifuged for 5 min at 2000 rpm to remove nonresuspendable debris, aliquoted and frozen to −80° C.

Transduction of Cells in Culture

HeLa cells (ATCC) were transduced by adding vector supernatants, whichmay or may not have been ultracentrifuged, in the presence of 10 μg/mlof DEAE dextran. The HeLa cells were cultivated in DMEM medium (ICN)supplemented by 10% foetal calf serum (FCS). HeLa cells were spread inan amount of 20000 cells/well onto a 96 well tray the day beforeinfection then transduced in a final volume of 200 μl. The vectorinnoculum was normalised to the concentration of capsid protein (P24),calculated using a commercial ELISA test (DuPont). The gene transferefficiency was measured using the experimental data for 24 to 48 hourspost infection. The amount of transduction of vectors expressing thelacZ reporter gene was revealed either by staining with Xgal in situ(Charneau et al, 1992), or by using a luminometric reaction using acommercial kit (Boehringer) following the manufacturer's instructions.In the case of vectors expressing the reporter gene EGFP, the amount oftransduction was qualitatively evaluated by direct observation of theliving cells using a fluorescence microscope, on the FITC channel. Thenumber of cells expressing the EGFP marker was quantified by flowcytometry (FITC channel). The EGFP protein was assayed by measuring thefluorescence of the cellular extract. The 96 well culture plates wererinsed twice with PBS then lysed with 100 μl of 1% NP40 PBS. The EGFPfluorescence was read using a plate fluorimeter (Victor, Wallac) with a475 nm excitation filter and a 510 nm emission filter.

HeLa cells stopped in their cell cycle in G1/S transition were preparedunder the same conditions as before with prior treatment 24 hours beforewith transduction with 4 μM of aphidicoline (Sigma). Under theseconditions, tritiated thymidine incorporation was inhibited by more than95%.

Ex vivo Transduction of Primary Cells

Primary rat spinal cord cells were prepared as follows: cords from 13 to14 day old rat embryos were dissected under a binocular magnifyingglass. The tissues were kept in L15 medium (Gibco) supplemented with 3.6mg/ml of glucose during all the steps. The nerve cells were dissociatedby incubating in trypsin (0.05% v/v) for 15 min at 37° C. Trypsicdigestion was inhibited by adding 10% foetal calf serum (FCS) andcentrifuging at low speed. The cellular pellet was taken up in L15medium, 3.6 mg/ml of glucose containing 100 μg/ml of DnaseI (Boehringer)by gentle mechanical agitation. The cells were collected by low speedcentrifuging through a 4% (w/v) BSA pad.

Spinal cells were seeded onto 24 well plates containing 12 mm diameterglass coverslips coated with poly-DL-ornithine (6 μg/ml) and laminin (3μg/ml). The cell cultures were maintained in a neurobasal medium (Gibco)containing B27 supplement, 2% FCS, 0.5 mM of L-glutamine, 25 μM ofbeta-mercaptoethanol and 25 μM of L-glutamate. After 24 hours, thecultures were treated with 10 μg/ml of 5′ fluorodeoxyuridine to preventcolonisation of the culture by non neuronal cells.

In vivo Transduction of EGFP in the Rat Brain

Vectors expressing the marker protein EGFP were used for in vivoexperiments.

The brains of 5 week old OFA spague dawley rats were injected with 2 μlof HR.EGFP vector or TRIP.EGFP vector. Firstly, the rats were put tosleep by intraperitoneal injection of Imagene 500 (Rhône Merieux). Theinjections were made into the striatum of each hemisphere using astereotaxic guide, with a 5 μl Hamilton needle, at a rate of 2 μl/5 min.The rats were sacrificed one week or more after injection, by perfusionof PBS then 2% paraformaldehyde (PFA). The brains were then removed andcut to retain only the portion containing the injection point, visibleby the lesion left by the needle. Post fixing with 2% PFA was carriedout overnight, followed by cryoprotection with 20% then 30% sucrose. Thebrains were then covered with tissue-tek, frozen in solid CO₂ and storedat −80° C. 14 μm slices were made using a cryostat then observed with aconfocal microscope.

In vivo and in vitro Comparison of HR luc and TRIP luc Vectors

One day before transduction, 20000 HeLa cells per well were spread onto96-well plates. Transduction was carried out using the same quantity ofvector particles normalised to the P24 content of the preparations: 1 ngof P24 per well, in triplicate, in the presence of 10 μg/ml of DEAEdextran. Two days after transduction, the luciferase activity wasmeasured using a Promega kit (following the manufacturer's instructions)and a Wallac microplate measuring device (Victor).

The vectors were injected into the brain striatum of OFA spague dawleyrats and C57B6 mice. 2 μl of a HR luc or TRIP luc preparation containing25 ng of P24 was injected (n=4). The animals were sacrificed 4 dayslater, the striatum was removed and the luciferase activity was measuredusing the same technique as before, simultaneously measuring the totalprotein quantity (Pierce kit).

EXAMPLES

1. Fundamental Aspects. Nuclear Import of Pre-Integration Complex

HIV-1: Role of Central Triplex

The mechanism for reverse transcription of the HIV virus differs fromthat of oncogene retroviruses in that the plus strand (+strand) issynthesised in two distinct halves (FIG. 1). A downstream segment isinitiated at a central copy of the polypurine tract (cPPT),characteristic of lentivirus genomes. Synthesis of the upstream plusstrand is terminated after a discrete displacement of the strand at thecentre of the genome. Blocking the displacement of the strand by reversetranscriptase is governed by a new cis-acting sequence of the HIVgenome: the CTS (central termination sequence). The final product ofreverse transcription of lentiviruses is thus a linear DNA carrying acentral structure spanning the strand (central triplex) over about ahundred nucleotides (Charneau et al, 1994). Specific mutagenesis of cPPTor CTS can halt initiation or central termination of synthesis of theplus strand. In both cases, mutant viruses, where the DNA is deprived ofthe central triplex, are defective for replication.

Analysis of a replicative defect in initiation and central terminationmutants has shown that the replicative cycle of initiation mutants orcentral reverse transcription termination mutants aborts during aposterior step in synthesis of viral DNA and posterior to routing thereverse transcription complex towards the nuclear envelope. When thestructure of the viral DNA present in the infected cells is analysed, itis seen that the phenotypes of the initiation and termination mutantsare similar. In both cases, the global reverse transcribed DNA contentis not affected by mutations in cPPT or CTS. In contrast, anaccumulation of linear non integrated DNA is observed, along with verylittle integrated provirus or circles with 1 or 2 LTRs formed over thesame period. Nucleus/cytoplasm fractionation experiments and nucleuspermeabilisation experiments have then shown that these linear DNAmolecules are associated with the nucleus, but that their integrationand/or circularisation can only occur after dissolving the nuclearenvelope, which clearly indicates that the viral DNA of the mutants iskept outside this envelope. Further, nuclease attack experiments on thepurified nuclei of cells infected with DnaseI immobilised on gold beadsagain show an accumulation of mutant linear DNA on the cytoplasmicsurface of the nuclear membrane. Finally, precise quantification of theintegrative capacity of the linear DNA molecules provided or notprovided with a wild type central triplex have recently shown that thecentral triplex does not influence integration of linear DNA into aheterologous DNA target in vitro.

The replicative defect of viruses mutated for initiation or centraltermination of reverse transcription thus concerns the nuclear import oftheir pre-integration complex and more precisely the step fortranslocation through nuclear pores. Lentiviruses, in particular the HIVvirus, have developed an original strategy for reverse transcriptionwherein the aim is to create the triplex at the centre of non integratedDNA molecules, an indispensable determinant for entry of the viralgenome into the nucleus of an interphase cell. This mechanismdistinguishes lentiviruses from all other retroviruses wherein access ofDNA to the integration site depends on the disorganisation of thenuclear membrane during mitosis.

2. Generation of Lentiviral Vectors Containing Cis-Acting SequencesResponsible for Triplex Formation 2-1 Principle and Importance ofLentiviral Vectors

The generation of effective lentiviral vectors assumes knowledge of thedeterminants responsible for active nuclear import and thus infection ofnon mitotic cells.

The discovery of the involvement of the triplex in the nuclear import ofthe HIV-1 genome has important consequences for the construction ofeffective lentiviral vectors. It assumes conservation in the vectorconstruction of cis-acting sequences responsible for the formation oftriplex DNA during lentiviral reverse transcription. The vectorologicalapplication of this fundamental research consists of adding the centralcPPT-CTS region to lentiviral constructions so as to create the triplexDNA structure during reverse transcription of the vector genome. Itshould be noted that many attempts at constructing a lentiviral vectorhave been made, based on the same principle as vectors derived fromoncovirus (in general MoMLV), and have proved to be disappointing atleast as regards the infectious titre. These vectors are replacementvectors, i.e., the ensemble of the viral genome is deleted thenreplaced, between the two LTRs and the packaging sequence, by thereporter gene or the gene of therapeutic interest (Miller et al, 1989).According to the inventors, this type of construction is not optimal inthe case of lentiviral vectors because of the need for the centralcPPT-CTS region for nuclear import of the viral DNA. However, HIVvectors constructed on the same principle as retroviral vectors derivedfrom oncovirus, but pseudotyped by the highly fusiogenic envelope of thevesicular stomatitis virus (VSV-G) and concentrated byultracentrifuging, enable in vivo transduction of rat neurons (Naldiniet al, 1997; Blomer et al, 1997) and of the liver and differentiatedmuscle (Kafri et al, 1997). However, complementation experiments withhuman pulmonary epithelium xenografts with these HIV vectors coding forthe CFTR (cystic fibrosis) gene have proved to be very disappointing.The essential portion of the DNA vector in this tissue remains in theform of linear non integrated DNA, thus revealing a probable defect innuclear import (Goldman et al, 1997; see the section “Influence ofcentral triplex on the amount of nuclear import of vector DNA”).

2.2 Construction and Production of “Triplex” HIV Vectors

In order to test the importance of the triplex structure in a vectorsystem, the inventors took as a basis the constructions described byNaldini et al. In this system (FIG. 2), HIV vector particles areproduced by transient co-transfection of three plasmids: a packagingplasmid expressing the whole of the viral proteins with the exception ofthe HIV envelope, a plasmid expressing the VSV-G envelope and a plasmidvector, pHR-CMVlacZ, comprising the LTRs of HIV, the bipartite packagingsignal of HIV and a lacZ expression cassette. Firstly, the lacZ reportergene was replaced by a gene coding for a highly fluorescent version ofEGFP (E green fluorescent protein), more practical for in vivotransduction studies. The central region of the HIV-1 LAI genomecomprising the cis-acting cPPT and CTS sequences, responsible fortriplex formation, was amplified by PCR then inserted into the ClaIsite, in the vector construction pTRIP-EGFP. Insertion of the wild typetriplex in the correct orientation generated the pTRIP-EFGP vector; inthe reverse orientation (non functional), it generated the pTRIPinv-EGFPvector.

2-3 Rapid and Sensitive Test for Detecting a Helper Virus in LentiviralVector Preparations: Absence of Infectious Helper Viruses in “VectorSupernatants”

The production of vector particles from three independent plasmids witha minimum of homologous sequences could minimise the probability ofgenerating a helper virus which was capable of replication. Further, thepackaging construction had been deleted for the HIV envelope and for theensemble of the genes said to be accessory to replication (Vif, Vpr,Vpu, Nef). The packaged vector genome no longer contained HIV other thanthe 2 LTRs, the sequences necessary for packaging of the triplexsequence. However, each vector stock was tested for the presence ofinfectious helper viruses. MT4 cells were infected in triplicate, on amicroplate, overnight, then washed extensively and taken up into cultureagain for 5 days in order to amplify the innoculum. P4 indicator cells(HeLa CD4 LTR-lacZ) were then infected with MT4 cells and theirsupernatant for 3 days to detect the infectious particles produced.Finally, in situ Xgal staining was carried out. In this manner, anyinfectious particle produced was detectable in the form of a bluescintillation. This sensitive protocol could detect an HIV innoculum of0.25 pg of P24, i.e., about 3200 physical particles. Knowing that in thecase of HIV, a single particle in 1000 or even in 10000 is infectious,the protocol can probably detect a single infectious particle.

The vector supernatants were systematically deprived of infectious HIVparticles.

2-4 Effect of Triplex on Transduction Efficiency by Vectors in vitro

Firstly, (FIG. 3), the effect of inserting the central triplex on HeLacell transduction was measured. HeLa cells were infected with atransfection supernatant from a wild type central triplex vector (TRIPGM, a vector without this sequence (HR GFP) or with a mutant triplexsequence (TRIP GFP D). The D mutant was a cPPT mutant which preventedcentral initiation of the +strand and thus the formation of the centraltriplex (FIG. 3). Infections were carried out with supernatantscontaining the same quantity of particles normalised to the quantity ofcapsid protein P24.

GFP transduction in HeLa cells was increased in the presence of a wildtype triplex and dropped to the base level in the presence of a nonfunctional triplex.

This increase in titre could be quantified using the lacZ reporter gene(FIG. 4). These cells were transduced in triplicate by normalising withrespect to the quantity of P24 protein. Transductions were carried outusing cells blocked or not blocked in division with aphidicoline, whichblocks G1/S cells. The vectors used were HRZ (no triplex), TRIP Z (withtriplex), TRIP Z inv (the triplex sequence was in the reverse direction,non functional, and did not lead to formation of a central triplex).

FIG. 4A: The gains in transduction of βgal by vectors containing thetriplex was 6 to 10 times. It was lost when the triplex was not formed(TRIP Z inv).

FIG. 4B: The effect of triplex on transduction of βgal was independentof cell division: similar results were obtained with cells in divisionor blocked with aphidicoline.

Further, the same results were obtained with HeLa cells, when thepackaging plasmid used during production of the vector particles was orwas not deleted in the accessory genes Vif, Vpr, Vpu, Nef.

2-5 Effect of Triplex on Efficiency of Transduction by ex vivo Vectors

The impact of the triplex on EGFP transduction in primary non mitoticcells was then measured. Primary explants from rat spinal cord enrichedin neurons were transduced with ultracentrifuged vector supernatants.The transductions were carried out with less than 10 μl ofultracentrifuged vector, containing the same number of particles,normalised to the number of ng of P24 capsid protein.

FIG. 6: The vector with a triplex sequence transduced a much largernumber of rat primary spinal cord explant cells than the vector withouttriplex.

2-6 Impact of Triplex on in vivo Transduction in the Brain

The effect of triplex on in vivo EGFP transduction was then measured bydirect injection into the rat brain. The same volume (2 μl) of vectorsupernatant with or without triplex containing the same quantity of P24protein was injected into the striatum. While a large number oftransduced cells was detected in rats injected with the vector withtriplex (FIG. 7 a), it was only possible to detect a few cellsexpressing EGFP in the brains of rats injected with the vector withouttriplex, at the exact point of the injection, visible by the lesion leftby the needle.

In FIG. 7 b, the construction of the HIV vectors (with or withouttriplex DNA sequence) which express the reporter gene luciferase (HR Lucand TRIP.Luc) enabled the impact on gene transduction in the brain to beprecisely quantified. In vitro an increase by a factor of 8 was observedin HeLa cells (FIG. 7-b1). An analogous benefit was obtained afterdirect injection in vivo into the brain striatum of the rat (FIG. 7-b2)or mouse (FIG. 7-b3).

2-7 Impact of Triplex on Nuclear Import of Vector Genome

A test which enabled the ensemble of the forms of DNA vector in thetransduced cell to be followed over time was developed by the inventors:linear DNA, circles with 1 or 2 LTRs but also integrated provirus. Thistest is based on detecting viral DNA by Southern blot using a cleavingstrategy and a choice of probe enabling the different forms ofretroviral DNA to be differentiated (see FIG. 8). The total DNA of theinfected cells or cells transduced by the vectors was digested by one ormore restriction enzymes to detach an internal fragment, common to allforms of retroviral DNA or vector DNA present in the cells (linear nonintegrated DNA, circularised DNA with one or two LTRs and integratedprovirus). In the case of a vector, the enzymes selected were Eco NI andAva II. When using as a probe a fragment generated by PCR exactlyspanning the Eco NI site, several bands corresponding to the differentforms of DNA appear. The internal fragment enabled the total vector DNApresent in the cells to be calculated after quantification using aPhosphorimager. A 1.16 kb fragment corresponded to the distal fragmentof non integrated linear DNA, a further 3.3 kb corresponded to nonintegrated circles. After quantifying the signals with thePhosphorimager, the degree of nuclear import was indicated by thepercentage of viral DNA integrated and in the circular form (nuclearviral DNA) with respect to the linear cytoplasmic DNA. The firstpreliminary blots showed an intracellular DNA profile characteristic ofa lack of nuclear import in the case of vectors deprived of triplex orwherein the central region of the HIV-1 genome had been inserted inreverse. The intensity of the signal corresponding to linear DNA wasequivalent to that of the total signal DNA 48 hours post infection. Inother words, processing of the DNA vector was mainly blocked in the nonintegrated linear stage, and very few molecules were integrated (FIG.9). In contrast, in the case of vectors with a triplex, only a littlelinear DNA subsisted after 48 hours, indicating that the major portionhad been imported into the nucleus of the transduced cell, thenintegrated.

2-8 Study of the Effect of the Position of the DNA Triplex on VectorConstruction

All lentiviruses contain cis-acting cPPT and CTS sequences responsiblefor triplex formation during reverse transcription. In all cases, thistriplex was found within a few nucleotides of the centre of the linearDNA genome. This central position of the triplex could be important forthe optimum function of this determinant of translocation through thenuclear pore. With the vector constructions produced, the triplexsequence had been inserted just upstream of the transcriptional unit ofthe reporter gene. Depending on the size of the reporter gene, thistriplex was found at a greater or lesser distance from the centre of thelinear vector DNA genome. In the case of the reporter gene EFGP (0.7kb), the triplex is very close to the centre of the construction; whilein the case of lacZ (3.1 kb), it is further away (FIG. 2). In bothcases, the presence of the triplex induced a large gain in titre in thesupernatant vectors. Thus there exists a certain “flexibility” in theposition of the triplex on the vector genome. However, vectors codingfor EGFP have been clearly shown to be more effective than those codingfor lacZ. It is thus possible that an ideally positioned triplex canresult in an additional gain in titre. In order to test this hypothesis,the inventors undertook to clone, in the place of reporter genes, a bankof fragments of random size (partial Sau3A digestion), the sizedistribution of the cloned fragment being analysed before and aftertransduction of the target cells. If the central position of the triplexis important to its function, constructing a symmetrical vector withrespect to the triplex would be important. It is possible to overcomethis obstacle by inserting the transcriptional unit of the vector intothe U3 region. After reverse transcription, the transgene will beduplicated either side of the triplex before being integrated, thusproviding the triplex with a precisely central position.

2-9 In vivo Transfer in Different Differentiated Tissues

The capacity of “triplex” lentiviral vectors to efficiently and stablytransduce the affected differentiated tissues in various geneticdisorders was studied. The potential of these vectors in the brain, andin different tissues such as muscle, pulmonary epithelium and liver inthe rat or mouse was studied. Qualitative responses could be obtainedrelatively rapidly using the EGFP reporter gene. Quantitativemeasurements of the impact of the triplex on the degree of transductionof these tissues were possible using the reporter gene luciferase.Further, the capacity of these vectors to transduce totipotent stemcells of human haematopoietic tissue could be evaluated, either frompurified CD34+cells or from total cord blood cells.

2-10 High Efficiency Gene Transfer in Haematopoietic Stem Cells byTriplex HIV Vectors

Haematopoietic stem cells are very important targets for the treatmentof a large number of genetic disorders connected with the blood, withmuscular disorders or with neurological disorders, and with infectiousdiseases. The major difficulty for gene transfer by retroviral vectorsderived from oncovirus such as MoMLV into these cells is that they onlyrarely divide and that inducing mitosis by a cytokine treatment isgenerally accompanied by a loss of totipotency. FIG. 16 shows theresults of transduction of the GFP gene in CD34 stem cells by theTRIP-GFP vector showing expression of GFP in more than 85% of the cells.The efficiency of transduction of CD34 stem cells by the vector withouttriplex, HR-GFP, was very low (Miyishi H et al, Science 1999, 283, p682-6). Since the CD34 stem cells were transduced immediately aftertheir purification, their clonogenic capacity remained intact.

2.11 Use of Lentiviral Vectors with a Triplex Sequence for Transductionof Embryonic Cells:

Application to the Construction of Transgenic Animals or Modified CellLines

Retroviral vectors are potentially important tools for the constructionof transgenic animals via egg transduction (Rubenstein et al, 1986,PNAS, 83, p 366-368) or ES cells (Friedrich and Soriano, 1991, GenesDev. 5, p 1513-1523). Using lentiviral vectors can increase theefficiency of transduction of these totipotent cells. Our preliminaryresults for transduction of mouse embryo cells via the TRIP-GFP vectorshow a high efficiency of transfer of the GFP gene but also completeextinction of transcription of the GFP transgene. Certain viralsequences, in particular the primary binding site (PBS), are suspectedof intervening in this extinction of expression. In order to overcomethis obstacle, self-deleting vectors for these viral sequences, focussedon the CRE/Lox specific recombination system (Choulika et al, 1996, J.Virol. 70, p 1792-98) were constructed.

2.12 Immunogenic Composition with Prophylactic and/or TherapeuticApplications

A Novel Immunisation Strategy: Triplex Lentiviral Vectors

Introduction

The role of T lymphocytes in the antitumoral and antiviral response hasbeen documented in many murine experimental systems and also in man.Different vaccine strategies aim at inducing a protective cytotoxicresponse against tumours or infectious agents. Lentiviruses have thecapacity of crossing nuclear pores and as a result are much better celltransduction vectors. In vitro and/or in vivo cell transduction can leadto the presentation by such cells of epitopes of tumoral and/or viralantigens which as a result induce a specific cellular immunity. Forthese reasons, the inventors have studied the immunogenic capacity ofrecombinant triplex lentiviral vectors using either in vitro transduceddendritic cells or direct in vivo administration to “humanised” mice byexpression of HLA-A2.1 (Pascolo S et al, 1997). This “HLA-A2.1 pure” HHDmouse is the best animal model for studying the restricted HLA-A2.1cytotoxic response and has been proposed for carrying out preclinicalimmunotherapy studies. The whole of our results clearly shows theimmunogenic capacity of triplex lentiviral vectors containing tumoralepitopes and thus triplex lentiviral vectors represent a novelimmunotherapeutic strategy.

Initial Study: Comparison of Different Vaccine Strategies

Different vaccine strategies were initially compared using HHD mice. Byarbitrarily selecting 5 tumoral epitopes, the inventors compared fiveimmunisation strategies applicable for human clinical practice: (i)synthetic peptides in incomplete Freund's adjuvant; (ii) lipopeptides(iii) recombinant yeast Ty particles in which, independently, theepitopes were fused at the C terminal end to the P1 protein; (iv)intramuscular administration of naked DNA coding the glycoprotein of thehepatitis B virus fused to epitopes in its pre-S2 portion; (v)intravenous injection of dendritic cells charged with peptides afterexpansion and differentiation in vitro from marrow cells. Havingobserved that the injections of particular structures (recombinant yeastTy) or naked recombinant DNA coding an S glycoprotein of the hepatitis Bvirus (refer to International patent application WO-A-95/11307,published 25, Apr. 1995) were the most effective strategies for inducingcytolytic responses, by inserting a polyepitopic moiety derived frommelanoma (10 distinct epitopes) into this glycoprotein, the inventorshave documented the possibility of simultaneously inducing cytolyticresponses against 5 epitopic peptides out of 10 in all of the test mice.

The particular Ty or naked DNA antigens proved to be effectivestrategies for inducing cytotoxic responses. However, the large scaleproduction of Ty particles is difficult. Further, it is feared thatintroducing multiple hydrophobic epitopes into the pre-S2 segment of thehepatitis B virus glycoprotein will entrain a large reduction in theproduction of particles by CHO cells (the current method for preparing ahepatitis vaccine). The recombinant lentiviruses (HIV-1) produced in theform of largely deleted pseudotypes but conserving the triplex DNAsequence have the capacity to traverse the nuclear membrane of nondividing cells and represent a novel vaccine strategy which ispotentially more effective with respect to the vaccine strategies citedabove.

Method, Apparatus and Results Transgenic Mice

HHD mice express a monocatenary construction in which the peptidepresentation (a1, a2) domains of the HLA-A2.1 molecule are covalentlyassociated at the N-end to the human β-2 microglobulin. The a3 domainand the intracytoplasmic portion of the HLA-A2.1 molecule were replacedby their equivalent in the H-2D^(b) molecule (Pascolo S et al, 1997).These mice enabled the immunogenicity of epitopic peptides and thedifferent vaccine strategies to be studied and compared.

Construction of TRIP-MEL IRES GFP Vector

Firstly, a bicistronic TRIP-IRES-GFP vector was constructed. The EcoRIsite of the TRIP-IRES-GFP vector was filled with T4 DNA polymerasecreating the TRIP-deltaE-GFP vector. Then a fragment of about 1.2 kb,BamHI-BstXI-SnaBI-EcoRI-IRES-EGFP-Xhol, was cloned in the place of theBamHI-EGFP-Xhol fragment. The fragment containing the IRES-EGFP(internal ribosome entry site) was generously donated by Dr YongwhonChoi (Rockefeller University, NY, USA). A fragment containing a Kozacconsensus sequence and a melanoma CTL polyepitope was generated by PCR,using the pBS meI poly matrix with pfu polymerase and the followingoligonucleotides: 5BgImlu Mel: 5′ cc aga tct acg cgt gcc acc atg gct gctggt 3′; 3RIMeI: 5′ CG GAA TTC GAC CTA AAC GCA ACG GAT G 3′. The meI PCRfragment was then digested with BgIII and EcoRI and cloned to the BamHIand EcoRI sites of the TRIP-deltaE-IRES-GFP vector creating theTRIP-MEL-IRES-GFP vector.

In vitro Transduction Efficiency of Dendritic Cells (DC) by GFPLentiviral Vectors with or without Triplex

Murine DCs were obtained from the marrow of transgenic HHD mice in thepresence of IL4 and GM-CSF. Human DCs were obtained from healthyHLA-A2.1 haplotype donors (see below). These cells were transduced by LVvectors with or without triplex using different concentrations (75, 150and 300 ng P24 of lentiviral vector per 5×10⁵ cells).

The expression of GFP in the DCs was measured by FACS on days 2, 5 and10. The values in terms of the average fluorescence intensitycorresponding to the cell transduction efficiency have shown thattriplex lentiviral vectors have 5 to 7 times the transduction capacityfor human DCs compared with lentiviral vectors without triplex.

Induction of Primary CTL Responses Using Human Dendritic CellsTransduced by the TRIP-MEL-IRES-GFP Vector

Immature human DCs were obtained from healthy HLA-A.2.1 haplotype donorsin the presence of GM-CSF and IL13 (IDM, Paris, France).Immunophenotyping of these cells by monoclonal antibodies against CD1,CD4, HLA-ABC, HLA-DR, CD80, and CD86 showed their immature nature with aDC purity of more than 91%.

The DCs obtained were transduced by the TRIP-MEL-IRES-GFP vector in aconcentration of 100 ng P24/vector per 1×10⁶ cells. The efficiency of DCtransduction by TRIP-MEL-IRES-GFP was studied by measuring theexpression of GFP by FACS. Mononuclear cells (MNC) from the same donorwere stimulated by the previously transduced DCs. After threestimulations, the cytotoxic activity of these cells was tested on T2cells individually charged with 4 epitopic peptides using a conventional4 hour CTL test. The epitopic peptides Mage-3, gp100.154, GnTV/NA17/A,and tyrosinase 368-D were selected because of their high immunogenicityin the previous experiments.

Specific cytotoxic responses were observed against all of the epitopestested. The lysis percentage for each epitope is shown in FIG. 12.

Direct Immunisation of HHD Mice by the TRIP-MEL-IRES-GFP Vector

HHD mice were immunised by 2.5 μ/P24 of the TRIP-MEL-IRES-GFP vector permouse subcutaneously (SC), intravenously (IV) and intraperitoneally(IP). On immunisation day 11, spleen cells from each mouse wereindividually stimulated by epitopic melanoma peptides for 6 days wherein2 days were in the presence of 10% TCGF. The lytic activity of thesecells was then tested on RMAS cells charged with the correspondingpeptides or on HeLa-HHD cells transduced by the TRIP-MEL-IRES-GFPvector.

The results obtained for each mouse are represented in terms of thespecific lysis of RMAS cells (Table 1) and of transduced HeLa-HHD cells(Table 2). The best results were obtained after administering the vectorby SC and IP mutes both in terms of lysis and the number of responsessimultaneously induced in a given mouse. The remarkable fact is that themajority of mice immunised by the IP route developed cytolytic responsesagainst all the epitopic peptides (FIG. 13).

TABLE 1 Specific cytotoxic response after immunisation withTRIP-MEL-IRES-GFP vector Specific lyses obtained after immunisation ofHHD mice by LV containing a melanoma polyepitope. In vitro individual SCstimulation for each mouse on day 8 in the presence of TCGF and peptideMouse gp154 gp209 gp280 gp457 G-nTV Mage-3 Mart 1.27 Mart1.32 Tyro-1Tyro368-D SC 1 25 4 13 8 54 17 17 4 4 10 2 44 1 5 11 89 10 11 4 3 6 3 390 19 21 81 29 26 6 4 0 IV 1 7 7 1 8 25 5 8 3 4 3 2 10 8 0 13 70 13 16 59 14 3 24 6 5 5 65 15 16 3 11 10 4 5 3 13 12 14 10 5 0 3 0 IP 1 30 10 23 57 9 6 4 3 2 2 63 8 7 17 72 11 19 9 7 7 3 21 7 8 16 72 14 32 0 7 7Target cells: RMAS charged with corresponding peptides Effector/targetratio: 30

Cytolytic responses f HHD mice immunised by SC, IV or OP routes with theTRIP-MEL-IRIS-GFP vector. Results obtained for HeLa-HHD cells expressingthe melanoma polyepitope.

TABLE 2 Specific cytotoxic response after immunisation withTRIP-MEL-IRES-GFP vector gp154 gp209 gp280 gp457 G-nTV Mage-3 Mart 1.27Mart1.32 Tyro-1 Tyro368-D SC 2 18 15 24 62 15 20 12 20 18 IV 8 10 15 2350 14 29 10 10 18 IP 24 18 15 25 62 15 32 14 18 20 Target cells:HeLa-HHD transduced by the TRIP-MEL-IRES-GFP vector Effector/targetratio: 30

CONCLUSION

The results demonstrate the capacity of triplex lentiviral vectors toinduce highly effective immune responses. Their immunogenic power hasbeen demonstrated not only in vitro on human dendritic cells but hasalso been evaluated in the transgenic HLA-A2.1 mouse using differentmodes of administration. Remarkably, specific CTL responses have beenobtained for the ten CTL epitopes contained in the melanoma polyepitope.The lysis percentages against melanoma antigens were also higher thanthose obtained with the same HHD mice with other vaccine strategies suchas lipopeptides, recombinant vaccine or DNA vaccination with HBVpseudo-particles. As a result, vaccine strategies based on triplexlentiviral vectors are applicable to a variety of tumoral or infectiousdisorders.

1-69. (canceled)
 70. A method for inducing an immune response against anexpressed transgene comprising administering lentiviral vector particlescomprising a replication-defective lentiviral vector that does notencode functional Gag, Pol, and Env proteins, to an animal, wherein thevector comprises: a transgene under the control of sequences regulatingtranscription; regulatory signals for reverse transcription andpackaging, and a lentiviral cis-acting central initiation region and alentiviral cis-acting termination region, such that a triplex DNA isformed during reverse transcription of the vector in the animal; andwherein the vector induces an immune response in the animal against theexpressed transgene.
 71. The method of claim 70, wherein the lentiviralcis-acting initiation region and the lentiviral cis-acting terminationregion are derived from HIV-1.
 72. The method of claim 70, wherein thelentiviral vector comprises HIV-1 regulatory signals for reversetranscription and packaging.
 73. The method of claim 71, wherein thelentiviral vector comprises HIV-1 regulatory signals for reversetranscription and packaging.
 74. The method of claim 70, wherein thelentiviral vector particles comprise HIV-1 Gag and Pol proteins.
 75. Themethod of claim 71, wherein the lentiviral vector particles compriseHIV-1 Gag and Pol proteins.
 76. The method of claim 72, wherein thelentiviral vector particles comprise HIV-1 Gag and Pol proteins.
 77. Themethod of claim 73, wherein the lentiviral vector particles compriseHIV-1 Gag and Pol proteins.
 78. The method of claim 70, wherein thelentiviral vector particles comprise vesicular stomatitis virus Envproteins.
 79. The method of claim 70, wherein the animal is a human. 80.The method of claim 76, wherein the animal is a human.
 81. The method ofclaim 77, wherein the animal is a human.
 82. The method of claim 78,wherein the animal is a human.
 83. A method for transferring a transgeneinto a target cell comprising transducing a target cell with alentiviral vector particle comprising a replication-defective lentiviralvector that does not encode functional Gag, Pol, and Env proteins, to ananimal, wherein the vector comprises: a transgene under the control ofsequences regulating transcription; regulatory signals for reversetranscription and packaging, and a lentiviral cis-acting centralinitiation region and a lentiviral cis-acting termination region, suchthat a triplex DNA is formed during reverse transcription of the vectorin the target cell.
 84. The method of claim 83, wherein the lentiviralcis-acting initiation region and the lentiviral cis-acting terminationregion are derived from HIV-1.
 85. The method of claim 83, wherein thelentiviral vector comprises HIV-1 regulatory signals for reversetranscription and packaging.
 86. The method of claim 84, wherein thelentiviral vector comprises HIV-1 regulatory signals for reversetranscription and packaging.
 87. The method of claim 83, wherein thelentiviral vector particle comprises HIV-1 Gag and Pol proteins.
 88. Themethod of claim 84, wherein the lentiviral vector particle comprisesHIV-1 Gag and Pol proteins.
 89. The method of claim 85, wherein thelentiviral vector particle comprises HIV-1 Gag and Pol proteins.
 90. Themethod of claim 86, wherein the lentiviral vector particle comprisesHIV-1 Gag and Pol proteins.
 91. The method of claim 83, wherein thelentiviral vector particle comprises vesicular stomatitis virus Envproteins.
 92. The method of claim 83, wherein the target cell is a humancell.
 93. The method of claim 89, wherein the target cell is a humancell.
 94. The method of claim 90, wherein the target cell is a humancell.
 95. The method of claim 91, wherein the target cell is a humancell.